Project Summary Antimicrobial resistance is a global health emergency. Despite this, the number of new antibiotics in development is limited and there is a desperate need for new pipelines to identify novel antibiotic candidates. We propose to develop a yeast-based drug-screening platform to be used in high-throughput compound screens to identify molecules with antimicrobial activity, targeting the highest priority pathogens. The overall strategy is to engineer the model organism Saccharomyces cerevisiae so its growth depends on the expression of sets of enzymes transplanted from human pathogens. By introducing entire enzymatic pathways, focusing on those that are essential in bacteria, fungus and plants but not in humans, the resulting yeast strains will provide an easy system for high-throughput drug screening and the opportunity to identify compounds with high therapeutic indices. Moreover, each enzymatic activity in the transplanted pathway will represent a unique druggable target, increasing the chances of compound identification. The goal of the Phase I study is to produce a series of engineered yeast strains expressing a pathway with multiple unique enzymatic activities, focusing mainly on priority pathogens as identified by the NIAID and the World Health Organization. Once developed, these strains can be used in high throughput drug screening studies. !